This invention relates to novel physiologically active porcine peptides of the CNP family (C-type natriuretic peptides). More particularly, the invention relates to a peptide composed of 53 amino acid residues and derivatives thereof.
Peptides assignable to two different peptide families that are named "atrial natriuretic peptide (ANP)" and "brain natriuretic peptide (BNP)" bave recently been discovered from mammalian atria and brains as hormones or nerve transmitters that regulate the homeostatic balance of body fluid volume and blood pressure in vivo. The structures of those peptides, the meebanism of their biosynthesis, as well as their physiological actions have also been unravelied.
The first clue to the discovery of ANP was reported by de Bold et al. in 1981. Finding that significant diuresis occurred when a rat's atrial crude extract was injected intravenously into another rat, de Bold et al. reported the existence of a natriuresis promoting factor in the atrium (de Bold, A. J. et al., Life Sci., 28, 89, 1981). Kangawa et al. later isolated that factor from human atrium, unravelled its structure and named it "atrial natriuretic peptide (ANP)" (Kangawa, K. et al., Bioahem. Biophys. Res. Commun., 118, 131, 1984; Kangawa, K. et al., Nature, 313, 397, 1985). It has been foijrid that litiman ANP (hANP) as it occurs in the atrium is classified into three types, .alpha.-, .beta.- and .gamma.-types, according to the molecular weight; .alpha.-type hANP (.alpha.-hANP) is a single-stranded peptide that consists of 28 amino acids having a single S-S bond in the molecule; .beta.-type hANP (.beta.-hANP) is an antiparallel dimer having an S-S bond formed between the molecules of .alpha.-hANP; and .gamma.-type hANP (Y-hANP) is a high-molecular weight protein composed of 126 amino acids, with .alpha.-hANP being contained in the C-terminal portion. Further, cDNA for hANP has been isolated and the routes of biosynthesis of .alpha.-, .beta.- and .gamma.-hANP have been identified on the basis of analysis of that cDNA, leading to the conclusion that each of those three types of hANP is biosynthesized from a common precursor protein (Oikawa, S. et al., Nature, 309, 724, 1984).
It is already known that among the three types of hANP, .alpha.-hANP is chiefly secreted into blood.
Ever since the structure of hANP was first unravelled, the structures of ANPs derived from other mammals have also been studied.
And, today, the following knowledge is available: ANPs have similar amino acid sequences over a broad spectrum of mammals ranging from rodents to humans; .alpha.-type ANP (.alpha.-ANP) has the same amino acid sequence in higher mammals including humans, dogs and pigs; and .alpha.-type ANPs derived from rats and rabbits have entirely the same amino acid sequence as .alpha.-hANP except that the methionine residue in position 12 is replaced by an isoleucine residue (Oikawa, S. et al., Biochem. Biophys. Res. Commun., 132, 892, 1985; Forssmann, W. G. et al., Anat. Embryol., 168, 307, 1983).
The first ANP isolate was obtained from the atrium but later studies involving the preparation of anti-ANP antibodies and examination of their distribution in vivo have shown that ANP also occurs in the brain as well as in the atrium. Since ANP-containing neurons have been reported to occur in the hypothalamus and pontine tegmentum of the brain (Cantin, M. et al., Histochemistry, 80, 113, 1984; Saper, C. B. et al., Science, 227, 1047, 1985), it is speculated today that ANP may also work in the brain as a nerve transmitter that participates in the regulation of the cardiovascular system.
The physiological actions of ANP are diverse and are not limited to a marked natriuretic action alone; it has recently been found that it is capable of not only lowering the blood pressure but also suppressing the production of aldosterone from the adrenal cortex. It is therefore clear today that ANP as it is secreted from the atrium into blood not only works as a hormone that regulates the homeostatic balance of body fluid volume and blood pressure but that in the brain it also works as a nerve transmitter for the nerve system to regulate the homeostatic balance of body fluid volume and blood pressure.
Brain natriuretic peptide (BNP) was first isolated from porcine brain and identified by Sudoh et al. in 1988 (Sudoh, T. et al., Nature, 332, 78, 1988). The first BNP isolate (pBNP-26) is a peptide that consists of 26 amino acid residues having a single S-S bond in the molecule and although it is similar to ANP in structure, i.e., in terms of primary amino acid sequence and the mode of S-S binding (producing a ring structure composed of 17 amino acid residues), BNP is clearly distinguishable from ANP. As in the case of ANP, natriuretic and hypotensive actions have been verified for BNP, which has therefore been named "brain natriuretic peptide (BNP)". At a later time, pBNP-32 composed of 32 amino acid residues having 6 amino acids attached to the N-terminus of pBNP-26 was isolated from porcine brain (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 155, 726, 1988); from porcine atrium, a peptide named ".gamma.-BNP" which was composed of 106 amino acids was also isolated and identified (Minamino, N. et al., Bioahem, Bioshys, Res. Commun., 157, 402, 1988).
On the basis of these results, it has been found that the peptides of the BNP family are biosynthesized from precursors that are entirely different from ANP. Further, as of today, the eDNAs of human and rat BNPs have been isolated and the structures of precursors for those BNPs have also become clear (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 159, 1427, 1989; Kojima, M. et al., Biochem. Biophys. Res. Commun., 159, 1420, 1989).
As already mentioned, BNP was first isolated from the brain. It was later found that BNP was present in porcine brain in an amount ten times as much as ANP and that like ANP, BNP also occurred in the atrium (though in an amount of only 2-3% of ANP) to be secreted into blood (Minamino, N. et al., Biochem. Biophys. Res. Commun., 155, 740, 1988; Aburaya, M. et al., Bioahem. Biophys. Res. Commun., 165, 872, 1989). From these facts, it was found that like ANP, BNP worked as a nerve transmitter in the brain and also worked as a hormone to be secreted from the atrium into blood, in either case helping regulate the homeostatic balance of body fluid volume and blood pressure.
In short, the following observations have been obtained to date: at least two obviously different families (ANP family and BNP family) of natriuretic peptides occur in mammals; peptides of these families are not only secreted from the atrium into blood and work as hormones that regulate the homeostatic balance of body fluid volume and blood pressure; they are also biosynthesized in the brain, where they work as nerve transmitters for the nerve system to regulate the homeostatic balance of body fluid volume and blood pressure.
As exemplified by natriuretic peptide, not a single peptide but a plurality of peptides can participate in the regulation of a certain physiological action in vivo (e.g. homeostasis of body fluid volume and blood pressure) and opioid peptide, tachykinin and endothelin have so far been recognized as other examples of such peptides. It has been known that three different families exist for each of these peptides (Hollt, V., Trend Neuro Sci., 6, 24, 1983; Nakanishi, S., Physiol. Review, 67, 1117, 1987; Inoue, A. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2863, 1989). This had increased the possibility that aside from the natriuretic peptides so far been known to be assignable to ANP and BNP families, peptides that could be classified into a third family might exist. In this regard, the present inventors very recently succeeded in isolating and identifying a novel peptide of a third family of natriuretic peptides from porcine brain and named it "CNP (C-type natriuretic peptide)" (which is hereinafter abbreviated as "CNP-22"). CNP-22 is a peptide composed of 22 amino acid residues and, like ANP and BNP, it forms a ring structure having a single S-S bond in the molecule. As in the case of ANP and BNP, the ring structure is composed of 17 amino acid residues and the primary amino acid sequence forming this ring structure in CNP-22 is highly homologous to that in .alpha.-ANP and BNP-32. However, the C-terminal portion of CNP-22 has a largely different structure than in ANP and BNP. Stated more specifically, the C-terminal portions of ANP and BNP have a "tail" structure in which a few additional amino acid residues are attached to the cysteine residue forming the ring structure, whereas the C-terminus of CNP-22 is a cysteine residue at position 22, with no "tail" structure being present. From these facts, it has been found that in spite of its structural similarity to ANP or BNP, CNP-22 is a peptide that belongs to an obviously different family of natriuretic peptides. Further, it has been verified that when administered to rats, CNP-22 exhibits natriuretic and hypotensive actions as in the case of ANP and BNP; it has therefore been found that CNP-22 is a new peptide assignable to a third family of natriuretic peptides in vivo (Japanese Patent Application No. 105047/1990). However, CNP-22 occurs in such smaller amounts in the brain than ANP and BNP that as of today no detailed information has been obtained with respect to the mechanism behind the biosynthesis of CNP-22, its distribution in vivo and physiological actions.